The present invention relates to modified peptide derivatives.
Many therapeutic agents operate by interacting with enzymes or receptors to modify their activity in a therapeutically beneficial manner. In the search for novel therapeutic agents it is thus sensible to concentrate on molecules which possess some degree of structural and conformational analogy with the natural substrate or ligand. Very often the identity of the natural substrate or ligand is unknown, but in many cases it can be inferred that a polypeptide is involved. It is therefore desirable to focus on compounds based on naturally occurring peptide structures, which can be screened for efficacy in interacting with the enzymes and/or receptors of interest.
It is known that the interaction of proteins controls key aspects of cellular function. For example protein-protein interactions control the signalling process causing a cell to divide, the malfunctioning of which may lead to cancer and other proliferative diseases. It has previously been demonstrated that an isolated peptide having the same sequence as the active portion of a natural protein may be used to bind with the natural protein-binding partner, and to elicit the same biological response. Processes of this type are described, for example, in WO97/11174, WO96/14339, WO96/35715 and WO97/42222.
Previous studies by the applicant have revealed that a range of non-peptide molecules may be used to mimic the structure of specific peptides within the natural polypeptide binding partners of various proteins which naturally bind protein ligands, and which may be used to interact with those proteins. This work is further described in WO 99/64574.
Of particular interest to the applicant are novel cyclophilin binding ligands. Cyclophilins are ubiquitous proteins highly conserved during evolution. They are found in bacteria, fungi, plants and vertebrates, and are widely expressed in many tissues. At least eight different forms of human cyclophilins have been identified, ranging from 18 kDa to 150 kDa in molecular mass [G{hacek over (o)}thel et al, Cell Mol. Life Sci 1999, 55, 433–436].
Cyclophilin is the major intracellular receptor for the immunosuppressive drug cyclosporin A [Handschumacher et al, Science 1984, 226,544–547]. In particular, cyclosporin A acts as an inhibitor of T-cell activation and can prevent graft rejection in organ and bone marrow transplantation [Borel, Pharmac. Review, 1969, 41, 259–371]. Cyclophilin is believed to be responsible for mediating this immunosuppressive response.
In addition, cyclophilin is also known to catalyse the interconversion of the cis and trans isomers of the peptidyl-prolyl amide bonds of peptide and protein substrates [Takahashi et al, Nature 1989, 337, 473–375; Fischer et al, Nature 1989, 227, 476–478]. Indeed, cyclophilin has been reported to accelerate the isomerisation of peptidyl-prolyl bonds in protein folding [Fisher et al, Biochemistry 1990, 29, 2205–2212]. Several mechanisms have been proposed including catalysis by (i) formation of a tetrahedral intermediate, (ii) distortion, (iii) protonation of the amide nitrogen, (iv) desolvation, or (v) a solvent assisted mechanism.
In order to elucidate the binding site for cyclosporin A, X-ray crystallographic studies have been carried out on a number of cyclosporin A-cyclophilin complexes. For example, Kallen et al [Nature 1991, 353, 276–279] disclose the X-ray crystal structure of human recombinant cyclophilin complexed with a tetrapeptide and identify the specific binding site for cyclosporin A by means of NMR spectroscopy. It was further revealed that the prolyl isomerase substrate binding site is coincident with the cyclosporin A binding site. Such results help provide a structural basis for rationalising the immunosuppressive function of the cyclosporin A-cyclophilin system and may also be important in the rational design of improved immunosuppressive drugs.
Studies by Zhao et al [Biochemistry 1996, 35, 7362–7368] disclose high resolution structures of cyclophilin A complexed with dipeptides of Ser-Pro, His-Pro and Gly-Pro. A comparison of these cyclophilin complexes reveals that the dipeptide structures have the same molecular conformation and bind in a similar manner. Moreover, the side chains of the N-terminal amino acids of the dipeptides do not strongly interact with cyclophilin, implying a minor contribution to any cis-trans isomerisation activity, thus accounting for the broad catalytic specificity of the enzyme.
WO98/25950 (Guildford Pharmaceuticals Inc.) discloses that small proline-containing tetra- or pentapeptides have a high affinity for cyclophilin-type immunophilins. Similarly, X-rays studies by Kallen and Walkinshaw [FEBS Letters, 1992, vol. 300, no. 3, 286–290] disclose the structure of a tetrapeptide bound to the active site of cyclophilin A, whereas Gallo et al [Biopolymers 1995, 36, 273–8] disclose binding experiments of cyclolinopeptide A [cyclo(-Pro1-Pro2-Phe3-Ph4-Leu5-Ile6-Ile7-Leu8-Val9)] with cyclophilin A.
In view of the properties described above, cyclophilin binding ligands are likely to be medically useful as inhibitory drugs. Indeed, the recent discovery that inhibition of cyclophilin prevents its incorporation into the HIV protein coat suggests that families of inhibitors unrelated to the immunosuppressant cyclosporins may provide potential anti-HIV drugs. Moreover, the development of species-specific cyclophilin inhibitors may also provide a route to novel anti-parasitic drugs.
The link between cyclophilin A and HIV has been the subject of a recent publication by Braaten D. and Luban J. [EMBO J Mar. 15, 2001;20(6):1300–9] which confirms the role of cyclophilin A in the regulation of the infectivity of HIV-1 virions.
Cyclophilin has also been implicated in certain types of cancer. For example, cyclophilin 40 is known to be overexpressed in breast tumours, compared to normal breast tissue [Breast Cancer Research and Treatment, 58, 267–280]. Estradiol treatment over a period of 24 hours has been shown to lead to a 5-fold increase in the expression of cyclophilin 40 mRNA in MCF-7 breast cancer cells [Biochemical and Biophysical Research Communications, 2001, 284, 219–225]. Further studies have revealed that allelic loss is detected in 30% of breast carcinomas from patients heterozygous for the cyclophilin 40 marker, suggesting that deletions of the cyclophilin 40 gene might be a late event in breast tumour progression [Journal Of Cancer Research And Clinical Oncology, 2001, 127, 109–115]. Cyclophilin 40 is also believed to play a role in liver cancer [Carcinogenesis, 2000, 21, 647–652].
Cyclophilin B has also been implicated in cancer. In this regard, Gomi et al have reported that the cyclophilin B gene encodes antigenic epitopes recognised by HLA-A24-restricted and tumour specific CTLs [Gomi S, Nakao M, Niiya F, Imamura Y, Kawano K, Nishizaka S, Hayashi A, Sobao Y, Oizumi K, Itoh K., J Immunol Nov. 1, 1999;163(9):4994–5004]. Tamura et al have identified a number of cyclophilin B-derived peptides capable of inducing histocompatability leukocyte antigen-A2-restricted and tumour-specific cytotoxic T lymphocytes [Tamura M, Nishizaka S, Maeda Y, Ito M, Harashima N, Harada M, Shichijo S, Itoh K., Jpn J Cancer Res, July 2001;92(7):762–7]. The present invention seeks to provide novel compounds which are capable of either binding to or inhibiting cyclophilin.